CN105838810B - A kind of screening technique of spot thatch breeding material - Google Patents
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Abstract
The present invention provides a kind of screening techniques of spot thatch breeding material, comprising the following steps: collects spot thatch resource, and carries out correlation analysis between the analysis of variance and character of phenotypic character to it；The genomic DNA of spot thatch is extracted as template, is expanded using SCoT labeled primer, polypropylene electrophoresis detection amplified production, DNA fingerprinting is obtained；Polymorphism analysis and the analysis of J genetic similarity index are carried out to spot thatch resource, and construct UPGMA genealogical tree；Comprehensive analysis is carried out according to the different ecological position of different spot thatch resources, phenotypic variation feature and genealogical tree, obtains screening material.Using technical solution of the present invention, analysis of variance and the molecular marking technique of phenotypic character is used in combination, so that the selection result is sensitiveer, accurate；The overall merit Exploitative potential of spot thatch germ plasm resource, has filtered out spot thatch breeding material, is cane breeding service.
Description
Technical field
The invention belongs to agricultural breeding technical field more particularly to a kind of screening techniques of spot thatch breeding material.
Background technique
Sugarcane is the most important industrial crops of the most important sugar crop in China and Guangxi.Modern Sugarcane Breeding is main
It is sweet in many decades " noble qualityization " breeding process with three kinds of blood relationships such as noble cane, minutia extraction (S. spontaneum) and India kinds
Sugarcane hereditary basis becomes gradually narrow.Therefore, go into overdrive to widen Sugarcane genetic basis using sugarcane wild resource be considered as
Cane breeding obtains one of critical path of breakthrough.
Spot thatch is the important wild germplasm of sugarcane, have many advantages, such as drought resisting, it is resistance to it is lean, waterlogging, disease-resistant, insect resistace is strong, at present
It is the hot spot of each sugarcane research institution primary study in the world.Traditional breeding selection is selected the variation of phenotypic character,
But some important characters can not come out in early detection, and Phenotypic Selection is easy the interference by environment.Molecular marking technique can
Analyzed according to genome genetic polymorphism from DNA angle is carried out, the accuracy of selection can be improved, breeding is shortened
Period improves the reliability of breeding efficiency and selection, quite easy.
Spot thatch has phenotypic character abundant, carries out phenotypic variation individually to analyze the Exploitative potential of spot thatch, is easy by ring
Border influences and finally influences spot thatch Exploitative potential.However, currently carrying out usability evaluation to spot thatch germ plasm resource, often only carry out
The investigation and analysis of phenotypic character, without the genetic analysis in phenotypic variation result binding molecule level is combined, also without
Method obtains the information such as the biggish spot thatch material of genotypic difference.
Summary of the invention
Against the above technical problems, the invention discloses a kind of screening technique of spot thatch breeding material, by phenotypic character with
Molecular marking technique is combined and is analyzed, and can efficiently use spot thatch germ plasm resource.
In this regard, the technical solution adopted by the present invention are as follows:
A kind of screening technique of spot thatch breeding material, comprising the following steps:
Step S1: spot thatch resource is collected, and carries out correlation analysis between the analysis of variance and character of phenotypic character to it；
Step S2: spot thatch genomic DNA is extracted as template, is expanded using SCoT labeled primer, polypropylene electrophoresis
Amplified production is detected, DNA fingerprinting is obtained；
Step S3: polymorphism analysis is carried out to spot thatch resource and genetic similarity index is analyzed, and constructs UPGMA system
Tree；
Step S4: comprehensive point is carried out according to the different ecological position of different spot thatch resources, phenotypic variation feature and genealogical tree
Analysis, obtains screening material.
As a further improvement of the present invention, in step S2, further include being screened to SCoT primer, filter out polymorphism
Spot thatch special marker primer that is high, being evenly distributed and concentrate the region 250bp -1000bp, is carried out using spot thatch special marker primer
Amplification.
As a further improvement of the present invention, the spot thatch special marker primer include SCoT1 primer, SCoT2 primer,
SCoT24 primer, SCoT25 primer, SCoT26 primer, SCoT27 primer, SCoT28 primer, SCoT29 primer, SCoT30 primer,
At least one of SCoT31 primer, SCoT35 primer, SCoT36 primer, SCoT37 primer, SCoT39 primer, SCoT42 primer；
Wherein, the amino acid sequence of the SCoT1 is SEQ ID NO:1, and the amino acid sequence of the SCoT2 is SEQ ID NO:2, institute
The amino acid sequence for stating SCoT24 is SEQ ID NO:3, and the amino acid sequence of the SCoT25 is SEQ ID NO:4, described
The amino acid sequence of SCo26 is SEQ ID NO:5, and the amino acid sequence of the SCoT27 is SEQ ID NO:6, the SCoT28
Amino acid sequence be SEQ ID NO:7, the amino acid sequence of the SCoT29 is SEQ ID NO:8, the ammonia of the SCoT30
Base acid sequence is SEQ ID NO:9, and the amino acid sequence of the SCoT31 is SEQ ID NO:10, the amino acid of the SCoT35
Sequence is SEQ ID NO:11, and the amino acid sequence of the SCoT36 is SEQ ID NO:12, the amino acid sequence of the SCoT37
It is classified as SEQ ID NO:13, the amino acid sequence of the SCoT39 is SEQ ID NO:14, the amino acid sequence of the SCoT42
For SEQ ID NO:15.
As a further improvement of the present invention, in step S3, when the genetic similarity index is analyzed, it is with similarity factor
0.73 is cut.
As a further improvement of the present invention, in step S2, reaction system when amplification is 20 μ l, wherein containing 10 ×
buffer(Mg2+) 2.0 μ l, 0.5 μ l of 50ng DNA profiling, 20 μm of ol/L primers, 4.0mmol/L dNTP, 0.2 μ l concentration be 5U/
The Taq archaeal dna polymerase of μ l.
As a further improvement of the present invention, amplification program are as follows: 94 DEG C of initial denaturation 4min, 95 DEG C of denaturation 50s, 50 DEG C of renaturation
40s, 72 DEG C of extension 2min, 36 circulations, last 72 DEG C of extensions 8min.
As a further improvement of the present invention, in step S2, PCR product is carried out using 6% non-denaturing polyacrylamide gel
Electrophoretic separation.
Target initiation codon polymorphic molecular marker (start condon targeted polymorphism, SCoT)
It has been found to be a kind of effective ways for analyzing crop genetic diversity and clustering relationships, which is that one kind is based on translating
The target gene labelling technique in beginning site designs single primer, passes through according to the conservative of translation initiation site ATG flanking sequence
PCR amplification generates the dominant polymorphism mark for being biased to candidate functional gene area, since the primer length for labeled analysis is longer,
Annealing temperature is higher, and is a kind of label based on PCR, and therefore, which has not only had both simplicity, low cost, rich
Rich polymorphism and it is reproducible the advantages that, and target gene can also be marked.
Genome DNA fingerprint atlas emphasizes uniqueness, this requires to have by the bands of a spectrum that primer amplification comes out preferable more
State property and specificity could effectively identify crop germplasm resource.The technical scheme is that being received first from different ecological environments
Collect spot thatch germ plasm resource, first carries out correlation analysis between the analysis of variance and character of crop field comparative test progress phenotypic character；Afterwards
Extracting genome DNA is carried out to spot thatch germplasm using CTAB method, high using polymorphism, mature effective SCoT label combines poly- third
Alkene gel electrophoresis technology carries out electrophoresis detection to amplified production, filters out that polymorphism is high, is evenly distributed and area from a plurality of primer
Domain concentrates on primer 15 of 250bp~1000bp.Polymorphism analysis, the genetic similarity index of spot thatch germ plasm resource are carried out again
Analysis and genealogical tree creation analysis；Finally combine different ecological position, phenotypic variation feature and genealogical tree, comprehensive analysis spot thatch kind
The Exploitative potential of matter resource.
Compared with prior art, the invention has the benefit that
The analysis of variance of phenotypic character is individually carried out, often due to environment influences accuracy that is big and influencing result, and
SCoT label is the sensitive technology of detection property in itself, and using technical solution of the present invention, the variation point of phenotypic character is used in combination
Analysis and molecular marking technique, so that result is sensitiveer, accurate；The overall merit Exploitative potential of spot thatch germ plasm resource, filters out
Spot thatch breeding material is cane breeding service.
Meanwhile technical solution of the present invention is further by comprehensive point of different ecological position, phenotypic variation feature and genealogical tree
Analysis, filters out that ecological environment differs greatly, biomass is higher and the biggish 5 parts of spots thatch clone of genotypic difference is educated as sugarcane
Emphasis in kind utilizes material.
Detailed description of the invention
Fig. 1 is the clonal ScoT testing result figure of SCoT29 primer pair spot thatch of the present invention.
Fig. 2 is the clonal UPGMA dendrogram of 50 parts of spot thatches of the invention.
Specific embodiment
With reference to the accompanying drawing, preferably embodiment of the invention is described in further detail.
Operating process: spot thatch resource collection-crop field phenotypic analysis test-different manifestations character analysis of variance-difference
Correlation analysis-extraction spot thatch genomic DNA-SCoT amplification-polyacrylate hydrogel electrophoresis-statistical between phenotypic character
Analysis-acquisition DNA fingerprinting-spot thatch resource polymorphism analysis-UPGMA genealogical tree building and analysis-different ecological position
It sets, phenotypic variation feature and genealogical tree comprehensive analysis-emphasis are screened using material.
Specific method and process are as follows:
(1) 50 parts of spot thatch clone is acquired from Guangxi different ecological area, while utilizes the sea of GPS apparatus measures collection point
It pulls out and longitude and latitude, 50 parts as shown in table 1 for the clonal title of examination spot thatch and source.
Table 1 is for the clonal title of examination spot thatch and source
(2) investigation of dominant phenotype character variation and analysis
It is planted using barrel plant, RANDOMIZED BLOCK DESIGN, 3 repetitions.It plants on 2 2nd, 2014, investigated its strain October 30
The indexs such as height, stem diameter, spike length, fringe joint number analyze the clonal quantitative variability of spot thatch.Using SAS software to quantitative character
The coefficient of variation (CV) analysis is carried out, the discrete journey of 4 quantitative characters is described.
As shown in table 2, the clonal 4 quantitative variability amplitudes of 50 parts of spot thatches are between 12.71%-16.14%.
Plant height range is 219-404cm, average out to 337cm；Stem diameter range is 0.56-1.07cm, average out to 0.82cm；Spike length range is
40.2-80.4cm, average out to 59.7cm；Fringe joint number range is 11-21 sections, 17 section of average out to.Exist between different materials certain
Difference shows apparent Morphological Diversity.It is stem diameter that variation amplitude is maximum, and the coefficient of variation is up to 16.14%；Followed by fringe
It is long, the coefficient of variation 15.97%；Plant height and the fringe joint number coefficient of variation are respectively 13.12% and 12.71%.
2 Guangxi spot thatch clonal form signature analysis of table
Correlation analysis is carried out to 50 parts of Guangxi spot thatch materials, 4 quantitative characters of separate sources, the results are shown in Table 3,
Plant height and stem diameter, spike length are in extremely significant positive correlation, and related coefficient is respectively 0.4468,0.328；Stem diameter and fringe joint number are in extremely aobvious
It writes and is positively correlated, related coefficient 0.211 is positively correlated with spike length in significant, related coefficient 0.0612；Spike length and fringe joint number are in pole
It is significant to be positively correlated, related coefficient 0.4529.The result shows that stem diameter becomes increasing as spot thatch constantly grows tall；Spot thatch
Plant height is higher, and flower spike is also longer；Stem diameter is larger and the longer spot thatch of flower spike, fringe joint number are more.
Correlativity between 3 Guangxi spot thatch Morphologic Characters of table
Note: * indicates significant correlation；* indicates extremely significant correlation.
(3) spot thatch extracting genome DNA
The genomic DNA of each spot thatch material is extracted using CTAB.
(4) design of primers and screening
50 parts of spot thatch materials such as GUX01, GUX02, GUX03, GUX04, GUX05 are selected, 46 pairs of SCoT primers are carried out just
Step screening, polypropylene electrophoresis detection amplified production.15 pairs of primer amplifications have gone out more visible bands of a spectrum, and have good repetition
Property, wherein bands of a spectrum are preferable between molecular weight ranges 250bp -1000bp, and partial results are shown in Fig. 1.
15 primers are filtered out from 46 pairs of primers, and (it is required that polymorphism and specificity are good, bands of a spectrum are at 250bp -1000bp points
Cloth relatively concentrate and uniformly, amino acid sequence is as shown in table 4.
4 SCoT labeled primer sequence of table
| Code | Sequence | Code | Sequence |
| SCoT1 | CAACAATGGCTACCACCA | SCoT30 | CAACAATGGCTACCAGCC |
| SCoT2 | CCATGGCTACCACCGGCA | SCoT31 | GCAACAATGGCTACCACC |
| SCoT24 | ACCATGGCTACCACCGGG | SCoT35 | ACCATGGCTACCACCGCC |
| SCoT25 | CAACAATGGCTACCACCC | SCoT36 | ACCATGGCTACCACCGTC |
| SCoT26 | CAACAATGGCTACCAGCA | SCoT37 | ACGACATGGCGACCACGT |
| SCoT27 | ACGACATGGCGACCCACA | SCoT39 | ACGACATGGCGACCGCGG |
| SCoT28 | CAACAATGGCTACCACGT | SCoT42 | CCATGGCTACCACCGCAC |
| SCoT29 | ACCATGGCTACCACCGCA |
(5) acquisition of spot thatch DNA fingerprinting, polymorphism analysis and genealogical tree building
50 parts of spot thatch materials such as GUX01, GUX02, GUX03, GUX04, GUX05 are selected, extract its genomic DNA as mould
Plate is utilized respectively above 15 primers and is expanded, polypropylene electrophoresis detection amplified production (Fig. 1).
Reaction system is 20 μ l, wherein (the Mg containing 10 × buffer2+) 2.0 μ l, 0.5 μ l of DNA profiling (50ng), 20 μm of ol
L-1 primer, 4.0mmolL-1dNTP, 0.2 μ l Taq archaeal dna polymerase (5U/ μ l).Amplification program are as follows: 94 DEG C of initial denaturations
4min, 95 DEG C of denaturation 50s, 50 DEG C of renaturation 40s, 72 DEG C of extension 2min, 36 recycle, last 72 DEG C of extensions 8min.PCR product is adopted
It is separated by electrophoresis with 6% non-denaturing polyacrylamide gel.
In line with the principle of " in different primers, the same allele site, the more rich primer result of polymorphism is selected ",
336 bands of a spectrum are filtered out from 15 pairs of primer specials, range detects 336, site, wherein polymorphic in 250bp -1000bp altogether
284, site of property, Polymorphisms 83.93%.Each primer polymorphism luffing is 69.57%-100%, polymorphism highest
Be primer SCoT24, up to 100.00%.Polymorphism it is minimum be primer SCoT35, be 69.57%.The position of every primer detection
Points are 17-30, and average number of sites is 22, wherein polymorphic site 16-26, average out to 19；Total number of sites and more
The most primer of state property number of sites is SCoT27, and see Table 5 for details.
The polymorphism of 5 15 SCoT primers of table
In the 50 parts of materials participated in the experiment, genetic similarity is between 0.65-0.81, average out to 0.71.Genetic similarity
It is up to GUX09 and GUX10, is 0.88；Minimum genetic similarity is GUX10 and GUX30, is 0.61.It is similar according to heredity
Property coefficient is clustered with UPGMA, constructs 50 parts of clonal Molecular Phylogenetic trees of spot thatch (Fig. 2).It is cut in similarity factor about 0.73
When cutting, all materials to be tested can be divided into 4 monoids.Wherein, GUX45, GUX49, GUX50 are the Ith monoid；GUX48 is the IIth
Monoid；GUX38 is the IIIth monoid；45 parts of clones such as GUX01, GUX02, GUX03 are the IVth monoid.
6. different ecological position, phenotypic variation feature and genealogical tree comprehensive analysis and emphasis are screened using material.
It is found that GUX38, GUX45, GUX48, GUX49, GUX50 and majority of material of one system of self-contained from genealogical tree
Genotypic difference it is larger, these materials are mainly from hazard prevention Yongfu county, Pingxiang City In Guangxi Province, southern China, Hechi City, Guangxi melt water county
The village Xia Zhangping and City: A Case Study in Baise Tianyang County, the plant height of these materials are higher than average water between 292-358cm mostly
It is flat, thus the biomass of these spot thatch materials can in higher level (plant height and stem diameter be spot thatch biomass main influence because
Son).In conjunction with different ecological weather, biomass and genotypic difference comprehensive analysis, filter out GUX38, GUX45, GUX48,
GUX49, GUX50 etc. 5 excellent, and the emphasis that should be used as in cane breeding utilizes material.
The present invention is investigated by phenotypic variation, shows 4 quantitative variability coefficients such as plant height, stem diameter, spike length, fringe joint number
Between 12.71%-16.14%, the coefficient of variation of stem diameter is maximum, is 16.14%, followed by spike length, is 15.97%, most
Small is fringe joint number, is 12.71%, and the Exploitative potential of stem diameter is maximum.Correlation analysis shows that plant height and stem diameter, spike length are in pole
Significant to be positively correlated, related coefficient is respectively 0.4468,0.328.Stem diameter and fringe joint number are in extremely significant positive correlation, and related coefficient is
0.211.Stem diameter and spike length are positively correlated in significant, related coefficient 0.0612.Spike length and fringe joint number are in extremely significant positive correlation, related
Coefficient is 0.4529；The spot thatch genome DNA fingerprint atlas obtained by 15 pairs of primers, 336 bands of a spectrum, wherein polymorphic site
284, polymorphism 83.93% illustrates that Guangxi spot thatch has more rich genetic diversity；Progress UPGMA clustering, 50
Part Guangxi spot thatch clone genetic similarity range is 0.65-0.81, when similarity factor is 0.73, can be divided into 4 classes
Group, the Guangxi spot thatch clone of areal is gathered substantially shows certain regionality distribution rule in same monoid；Finally will
Different ecological position, phenotypic variation feature and genealogical tree comprehensive analysis, filter out that ecological environment differs greatly, biomass is higher and
The biggish 5 parts of spot thatch clones of genotypic difference utilize material as the emphasis in cane breeding.
From the above experiments, it was found that phenotypic variation is used in combination with SCoT label, so that result is effective and accurate.Sieve
The Appropriate primers for the 15 pairs of spot thatch germplasm selected, polymorphism and specificity are good, and bands of a spectrum are relatively concentrated, in 250bp -1000bp distribution
It is even.By different ecological position, phenotypic variation feature and genealogical tree comprehensive analysis, having filtered out can pay close attention in cane breeding
Spot thatch germplasm materials.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.
SEQUENCE LISTING
<110>Guangxi Autonomous Region Academy of Agricultural Sciences sugarcane research institute
<120>a kind of screening technique of spot thatch breeding material
<160> 15
<170> PatentIn version 3.3
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Claims (5)
1. a kind of screening technique of spot thatch breeding material, it is characterised in that: the following steps are included:
Step S1: spot thatch resource is collected, and carries out correlation analysis between the analysis of variance and character of phenotypic character to it；
Step S2: being extracted each spot thatch genomic DNA as template, expanded using SCoT labeled primer, the inspection of polypropylene electrophoresis
Amplified production is surveyed, DNA fingerprinting is obtained；
Step S3: polymorphism analysis is carried out to spot thatch resource and genetic similarity index is analyzed, and constructs UPGMA genealogical tree；
Step S4: comprehensive analysis is carried out according to the different ecological position of different spot thatch resources, phenotypic variation feature and genealogical tree, is obtained
To screening material；
Further include being screened to SCoT primer in step S2, filters out that polymorphism is high, be evenly distributed and concentrates 250 bp-
The spot thatch special marker primer in 1000 regions bp, is expanded using spot thatch special marker primer；
The spot thatch special marker primer includes SCoT1 primer, SCoT2 primer, SCoT24 primer, SCoT25 primer, SCoT26
Primer, SCoT27 primer, SCoT28 primer, SCoT29 primer, SCoT30 primer, SCoT31 primer, SCoT35 primer, SCoT36
At least one of primer, SCoT37 primer, SCoT39 primer, SCoT42 primer；Wherein, the amino acid sequence of the SCoT1 is
The amino acid sequence of SEQ ID NO:1, the SCoT2 are SEQ ID NO:2, and the amino acid sequence of the SCoT24 is SEQ ID
The amino acid sequence of NO:3, the SCoT25 are SEQ ID NO:4, and the amino acid sequence of the SCo26 is SEQ ID NO:5,
The amino acid sequence of the SCoT27 is SEQ ID NO:6, and the amino acid sequence of the SCoT28 is SEQ ID NO:7, described
The amino acid sequence of SCoT29 is SEQ ID NO:8, and the amino acid sequence of the SCoT30 is SEQ ID NO:9, described
The amino acid sequence of SCoT31 is SEQ ID NO:10, and the amino acid sequence of the SCoT35 is SEQ ID NO:11, described
The amino acid sequence of SCoT36 is SEQ ID NO:12, and the amino acid sequence of the SCoT37 is SEQ ID NO:13, described
The amino acid sequence of SCoT39 is SEQ ID NO:14, and the amino acid sequence of the SCoT42 is SEQ ID NO:15.
2. the screening technique of spot thatch breeding material according to claim 1, it is characterised in that: in step S3, the heredity
When likeness coefficient is analyzed, cut with similarity factor for 0.73.
3. the screening technique of spot thatch breeding material according to claim 1, it is characterised in that: in step S2, when amplification
Reaction system is 20 μ l, wherein containing 10 × Mg2+Buffer2.0 μ l, 50 ng DNA profiling, 0.5 μ l, 20 μm of ol/L primers,
4.0 mmol/L dNTP, the Taq archaeal dna polymerase that 0.2 μ l concentration is 5 U/ μ l.
4. the screening technique of spot thatch breeding material according to claim 3, it is characterised in that: amplification program are as follows: 94 DEG C pre-
4 min are denaturalized, 95 DEG C of 50 s of denaturation, 50 DEG C of 40 s of renaturation, 72 DEG C of 2 min of extension, 36 circulations, last 72 DEG C extend 8
min。
5. the screening technique of spot thatch breeding material according to claim 3, it is characterised in that: in step S2, PCR product is adopted
It is separated by electrophoresis with 6% non-denaturing polyacrylamide gel.
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| CN1566362A (en) * | 2003-07-10 | 2005-01-19 | 福建农林大学 | DNA identification method for hybrid of sugarcane and saccharum arundinaceum |
| CN103468790A (en) * | 2013-06-04 | 2013-12-25 | 四川省草原科学研究院 | Primers used for detecting polymorphism of saccharum arundinaceum, and detection method and applications |
| CN104673904A (en) * | 2015-02-11 | 2015-06-03 | 华南农业大学 | Sugarcane smut SCoT-PCR specific primer screening and application |
-
2016
- 2016-05-20 CN CN201610339595.2A patent/CN105838810B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1566362A (en) * | 2003-07-10 | 2005-01-19 | 福建农林大学 | DNA identification method for hybrid of sugarcane and saccharum arundinaceum |
| CN103468790A (en) * | 2013-06-04 | 2013-12-25 | 四川省草原科学研究院 | Primers used for detecting polymorphism of saccharum arundinaceum, and detection method and applications |
| CN104673904A (en) * | 2015-02-11 | 2015-06-03 | 华南农业大学 | Sugarcane smut SCoT-PCR specific primer screening and application |
Non-Patent Citations (2)
| Title |
|---|
| SCoT 分子标记在割手密遗传图谱构建中的应用;罗霆 等;《植物遗传资源学报》;20130607;第14卷(第4期);第704-710页 * |
| 基于SCoT标记分析甘蔗种质资源遗传多样性;杨翠;《中国优秀硕士学位论文全文数据库 农业科技辑》;20110415;D047-251 * |
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